Proliferation of vascular smooth muscle cells (VSMCs) is fundamental to neointima formation, a hallmark of cardiovascular disease (CVD). The extracellular matrix (ECM) plays an important role in regulating VSMCs, and we have studied the effects of the ECM component hyaluronan (HA) and the HA receptor, CD44, on VSMC proliferation and neointima formation. We show that HA and CD44 have bifunctional effects on VSMC proliferation and activation. In vitro, the native high molecular weight form of HA (HMW-HA) antagonizes mitogen-induced S phase entry of VSMCs while the lower molecular weight forms of HA (LMW- HA) that accumulate at sites of inflammation synergize with mitogens to stimulate VSMC S phase entry. Both effects are mediated by CD44, and result from differential regulation of Rac- and ERK-dependent signaling to cyclin D1. HMW-HA binding to CD44 inhibits GTP-loading of Rac and Rac-dependent signaling to the cyclin D1 gene while LMW-HA binding to CD44 stimulates ERK-dependent cyclin D1 gene expression. A similar bifunctionality can be detected in vivo as genetic deletion of CD44 enhances the response to injury in C57BL/6 mice but inhibits atherogenesis in apoE-deficient mice. Since the ratio of LMW-HA to HMW-HA is directly related to the extent of inflammation, and the inflammatory environment is much greater in apoE-deficient mice as compared to B6 mice, we hypothesize that the biological response to CD44 in vivo is directed by the degree of local inflammation, and that changes in the inflammatory milieu can reconcile the seemingly contradictory effects of CD44 on VSMC activation and neointima formation during the response to vascular injury and atherogenesis. In Aim 1, we will compare the effects of CD44 on VSMC proliferation and neointima formation in vascular injury as we modulate inflammation, and we will exploit our recently developed floxed CD44 mouse to distinguish the effects of CD44 on SMCs and inflammatory cells. In Aim 2, we will perform similar experiments monitoring the effect of inflammation and cell type-specific CD44 expression on neointima formation during atherogenesis. In Aim 3, we will attempt to eliminate the bifunctional response of CD44 on VSMC activation by performing fine-wire injury on mice carrying an SMC-specific deletion of Rac1. PUBLIC HEALTH RELEVANCE: Cardiovascular disease due to atherosclerosis, the number one cause of death in the United States, is characterized by neointima formation due to inflammation and vascular smooth muscle cell proliferation. The balance between inflammation and formation of smooth muscle cell-rich fibrotic caps in part determines the risk to rupture of atherosclerotic plaques and acute events such as heart attacks. Minimally invasive percutaneous revascularization procedures such as angioplasty and stent implantation are currently considered the treatment of choice for the prevention and treatment of acute coronary events in many patients. However, such interventions are associated with a significant risk of vascular restenosis due to excessive vascular smooth cell proliferation in response to injury caused by the intervention. Modulating smooth muscle cell proliferation may be an important approach to minimize the risk of acute events in patients with atherosclerosis, and may also prevent restenosis following physical interventions, thereby greatly reducing the risks associated with standard therapeutic interventions. The proposed studies will define mechanisms that regulate smooth muscle cell growth and thus provide novel targets for treatment of atherosclerosis and restenosis.